Class II major histocompatibility complex (MHC) gene products play critical roles in a variety of T and B lymphocyte responses. Biochemical and functional analyses have been used to investigate the relationship between class II structure and peptide antigen presentation. The kinetics of class II peptide binding have been examined and two distinct phases of binding identified; a rapid initial binding with a rapid off rate, and a slow accumulation of long-lived binary complexes. Low affinity binding was unexpectedly found to preserve class II molecules from denaturation at physiological temperature, a result with implications for the function of invariant chain (see Z01 AI 00545-05 LI). The direct demonstration of different affinities of interaction of class II with the same peptide suggests that optimal alignment of peptide side chain and class II binding pockets is an infrequent event that provides a kinetic limit to the rate of high affinity peptide capture by class II molecules. These new insights into the biochemical behavior of class II help provide a deeper understanding of how antigen capture by class II molecules occurs under physiological conditions. In addition to binding peptide and being recognized by clonally distributed T cell receptors, class II molecules participate in T cell selection in the thymus and mature T cell activation in the periphery by interacting with the CD4 molecule that is also the receptor for HIV-1. We have used site-directed mutagenesis to define the site(s) of interaction of class II molecules with CD4. In addition to our previous identification of a major binding site in the beta2 region of the class II molecules, we have now located a second important site in the alpha2 domain. These two sites on a single class II heterodimer cannot both bind to an individual CD4 molecule simultaneously. The two sites do lie close together in the recently published crystal structure of the class II "dimer of dimers". Taken together, these results provide the first evidence for organization of higher order oligomeric signal transduction complexes involving the T cell receptor and CD4 coreceptor. These latter findings, in concert with our studies of T cell signal transduction upon exposure to distinct peptide-MHC ligand complexes (see Z01 AI 00403-11 LI), are defining the molecular mechanisms involved in MHC-dependent antigen recognition by, and activation of T lymphocytes.